Many pathogenic viruses, against which vaccines would be desirable, are simply too proficient at producing disease to be administered in a live product from the naturally-occurring organism. Furthermore, even when a virus strain does not itself produce disease, it may have characteristics that, when recombined with those of another strain encountered in the field, result in a far worse disease outbreak. Such is the case with influenza viruses, which have a segmented genome capable of reassortment. If an attenuated vaccine strain with certain gene segments that give it the ability to replicate well in a given host species should exchange segments with another virulent strain that does not replicate well in that species, the outcome may be a new strain that is both virulent and well-adapted to the new host. That was the fear that existed at the time of the 1997 Hong Kong outbreak in humans of an avian strain of influenza. Fortunately, although the avian strain was able to colonize certain humans, it did not replicate well enough in that host to cause an epidemic in what would have been a completely susceptible, immunologically naive population.
Among the veterinary influenza products, there are eight avian vaccines manufactured by one company. One swine vaccine and five equine vaccines produced by six different companies. They are all killed virus products, as are the human influenza vaccines used in annual vaccination programs. Human vaccines are changed annually to reflect the newly emerging strains and frequently contain 3 distinct hemagglutinin antigens from diverse strains to maximize protection against disease.
One problem with inactivated influenza virus vaccines is that the immunity generated is only partial. In the presence of a strong adjuvant, antigens can stimulate B-cells and induce a good humoral response. However, there is little cell-mediated immunity generated by a killed product. This can mean the difference between disease and full or partial protection. Furthermore, the immunity provided by a killed product can be relatively short-lived. The potential advantages of a recombinant vaccine, when administered in a safe vector, are that it may express protective immunogens against even the most dangerous of viruses, provide both humoral and cell-mediated immunity, and extend the duration of that protection beyond the time provided by a killed product.
The pathology induced by SIV occurs throughout the respiratory tract and consists of acute inflammation, edema, and necrosis. More severe complications, in the form of interstitial pneumonia, thickening of alveolar walls, hyperemia, thrombosis, hemorrhage, and necrosis, can occur. Lung lesions tend to be bilaterally distributed, predominantly in the cranial and middle lobes. Generally, most pigs recover but resolution of lesions may take up to a month.
In recent years there have been periodic occurrences of `atypical` SIV outbreaks, leading to speculation that the relatively stable antigenic profile of SIV may be changing. Reports have appeared in the literature since 1992 which indicate regarding the occurrence of SIV either associated with unusual signs or exhibiting more virulence than expected. First, there was a report from Quebec regarding an H1N1 variant producing proliferative and necrotizing pneumonia in pigs. Some symptoms were very similar to those seen arising from infection with the PRRS virus. More point mutations and diversity were observed than are usually seen in North American SIV isolates. Another novel isolate from a severely affected herd was designated A/Sw/Nebraska/1/92. That strain induced persistent, high fevers (up to 42.degree. C.) but not much respiratory disease. Given the high degree of conserved sequences in typical SIV strains isolated in the U.S., it was surprising that the most closely related reference H1N1 strain had only 94% identity at the nucleotide level and 96% identity at the amino acid level to this SIV isolate. Nonetheless, it was closer genetically to `classical` H1N1 SIV than to avian or human H1N1 viruses. In England, an H1N1 strain antigenically distinguishable from classic SIV and European avian-like H1N1 viruses caused a sudden increase in SIV cases, but still resulted in the usual clinical signs of coughing, sneezing, and anorexia. However, upon experimental infection, this strain produced a more severe interstitial pneumonia and hemorrhagic lymph nodes.
The significance of the genetic diversity represented by these strains is as yet undetermined. It may be that there are no `atypical` SIV strains, merely a greater degree of potential antigenic diversity among field strains than previously detected. It is known, however, that subtype H1N1 influenza viruses have been circulating continuously in U.S. pigs for over 60 years. It is believed that the great pandemic of "Spanish flu" in 1918/19, the worst in history, killing at least 20 million people worldwide, was either caused by a swine virus or by a human strain that entered the pig population at that time. In 1997, RNA from a casualty was extracted from formalin-fixed, paraffin-embedded tissue and sequenced. All sequences determined were very similar to those of classic H1N1 SIV, suggesting that human and swine strains share a common avian ancestor, existing some time before 1918.
Northern Europe saw its first isolate of SIV in 1978/79, and although in H1N1 virus the H1 was similar to the avian H1, it is but distinct from both human and swine H1. Since then, there have been instances where an avian virus has been able to cross species and infect the pig population, as with SwGer/81. There are also and cases where reassortment between avian and classical SIV has occurred, such as is seen with SwHK/82. In addition, there is a human virus-like H3N2 subtype that has been isolated on occasion in European pigs since 1980. this may result from the 1968 antigenic shift and an ability of the H3N2 to persist in pigs even when not circulating in the human population. It is interesting that a serological survey conducted in 1988-1989 in U.S. pigs found evidence of H3 viruses antigenically similar to the human H3 strains, which was current at that time, at about 1.1% average prevalence. In addition, a serological survey during 1976-1977 detected an incidence of 1.4% for H3N2 infections. Moreover isolation from one herd of a virus antigenically similar to a human H3N2 strain was reported in that study. However, complete sequencing to determine whether the isolate was of human origin was not performed. However, no H3 human strain has been confirmed as present in U.S. pigs. Very recently, the National Veterinary Services Laboratories identified an influenza virus subtype H3N2 isolate from a swine breeding herd in North Carolina (personal communication). Studies are currently underway to determine species of origin and other characteristics of the virus.
In summary, it is recognized that there are at least three HA subtypes circulating in pigs at present, classic SIV H1, avian virus-like H1, and human virus-like H3. These have been found in various permutations of SIV gene segments. One, an H3N1 strain, appeared to be a combination of the classic SIV and the human virus-like H3N2 found in swine. Another, an H1N2 isolate believed to be from a human H1N1 and the swine-adapted H3N2, caused clinical disease in pigs. Still another represented a reassortment between human and avian strains in symptomatic Italian pigs, providing the first proof that pigs can act as `mixing vessels` for human and avian viruses.
The critical role that pigs can play in pandemics was underscored by the discovery that children in the Netherlands were sick from avian-human influenza virus generated in pigs, transmitted pig-to-person, and person-to-person. Normally, avian strains do not replicate in humans, and human strains do not replicate in birds. This is a function of their specific sialyloligosaccharide receptors on the surface of epithelial cells of the upper respiratory tract. In a previous study, it was determined that, of 38 avian influenza strains, fully 31 were successfully transmitted to swine. Every HA subtype (of 14 tested) had at least one strain that grew as well as a swine or human virus. Since then, it has been determined that pigs, in fact, have both avian- and human-specific viral receptors present in their upper respiratory tract and that some avian strains, with continued replication, acquire the ability to recognize human receptors as they become swine-adapted. Taken together, these data delineate the danger to humans of a pig population unprotected against influenza. If an avian virus with a non-human-type HA is introduced into pigs, then reasserts with a human strain, a pandemic among complete susceptibles would occur. Although direct interspecies spread from bird to human can happen, as was seen with the 1997 Hong Kong H5N1 cases, the virus under those circumstances may not readily adapt to its new host and relatively few may be affected. The dangers may be greater when interspecies transmission occurs with the pig, able to be infected by either avian or human strains serving as a `mixing vessel`, wherein gene segments from different strains reassort to produce new viruses. Clearly, a safe, live vaccine vector able to express multiple genes that could be given frequently to boost or provide new immunity,.would be helpful.
Vaccinia virus, a member of the Orthopoxviruses, is known to be a strong inducer of humoral and cell-mediated immunity. Vaccinia virus has been used as an experimental vector in several species and in accidental human exposure, with considerable documentation of its ability to induce immunity to several diseases. The first reports of its use as a vector for the delivery of immunogens surfaced in the early 1980's. By 1990, there were numerous reports in the literature regarding its successful expression of the hepatitis B virus surface antigen, the thymidine kinase and glycoprotein D from herpes simplex virus, the influenza hemagglutinin, human respiratory syncytial virus glycoprotein G, and the rabies virus glycoprotein. Several studies demonstrated its immunogenicity in humans. Insertion of more than one gene was also accomplished. For example, one recombinant expressing both the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D was produced, thus raising the possibility of using a single vector expressing immunogens from multiple pathogens, thereby providing protection against all of them with a single vaccine.
As promising as vaccinia virus seemed to be for certain applications, it had several drawbacks which limited its potential for general use. During the smallpox eradication effort, vaccinia caused unwelcome side effects. Apart from the localized irritation induced by the inoculation, there were more severe complications among the immunocompromised. It was estimated that one in every million vaccinations resulted in death. For this reason, vaccinia virus was no longer used clinically in the U.S. or the rest of the world following the end of smallpox vaccination. Following decades of little to no use, the general population, at one time well exposed, has now become quite susceptible, as people less than roughly 30 years of age have no immunity. Unfortunately, this risk of human exposure greatly curtails the usefulness of the naturally occurring vaccinia as a vector. Even if a vaccine were designed for some one species in the veterinary market, because vaccinia has such a wide host range, there exists the potential for inadvertent inter-species spread.
To circumvent the problems associated with vaccinia virus, yet retain its advantages, researchers have investigated strains of vaccinia virus that are attenuated, either by nature or design. One such modified strain, Ankara (MVA), has been widely studied. The strain was originally developed from the vaccinia virus Ankara strain as a safe alternative for smallpox vaccination, and has been used without significant side-effects in over 120,000 people, including young children and the elderly, for immunization against smallpox. After approximately 570 passages in primary chick embryo fibroblasts (CEF), it has lost its ability to replicate or at least to replicate well in numerous mammalian cell lines. It contains six major deletions that prevent virus assembly in almost all mammalian cells tested. However, gene expression, both early and late, remains relatively unimpaired. The exact nature of this host restriction is not really understood. Thus far, four orthopox virus host range genes have been documented. These are designated CHOhr, C7L, K1L, and E3L genes. Of these, only the function of the E3L gene, which expresses an RNA binding protein, is known. Compared to its parental strain, MVA has deletions that consist of about 15% (30,000 base pairs) of its former genome, including deletion of most of the K1L gene. Interestingly, one study showed, replacement of the K1L gene in MVA removed only the host restriction in RK13 cells. This suggests that there are multiple, cumulative genetic defects in MVA replication. If so, as seems likely the probability of spontaneous reversion to a wild type host range is quite low. It can be assumed, therefore, that the deletions greatly increase the safety of MVA for use as a vaccine vector.